TRPC current inhibition and PI(4,5)P₂ reduction in response to the protocol for measuring the voltage dependence of DrVSP activation. (A) TRPC6, CFP_mse-PHd, YFP_mse-PHd (PI(4,5)P₂ sensor), and voltage-sensing phosphatase (DrVSP) were coexpressed in HEK293 cells. Gradual current inhibition and reduction in PI(4,5)P₂ caused by the step-pulse protocol (left; from 20 to 180 mV; duration of 500 ms; repeated every 25 s). A DAG lipase inhibitor (RHC80267; 100 µM) was applied to induce the currents (gray horizontal bar). The ratio of current inhibition, r(I), and FRET reduction, r(FR), upon the depolarization pulses was used to quantify the channel activity and PI(4,5)P₂ changes after DrVSP activation (right). (B) The voltage dependence of current inhibition (left axis; circles) and FR reduction (right axis; triangles) after DrVSP activation in cells expressing TRPC3 (left), TRPC6 (middle), and TRPC7 (right) channels.